Abstract
| - His 238, a conserved amino acid located in hydrogen-bondingdistance from C-6 of the substratein the active site of murine adenosine deaminase (mADA) and postulatedto play an important role incatalysis, was altered into an alanine, a glutamate, and an arginineusing site-directed mutagenesis. TheAla and Glu substitutions did not result in changes of the secondary ortertiary structure, while the Argmutation caused local perturbations in tertiary structure and quenchedthe emission of one or more enzymetryptophans. Neither the Glu or Arg mutations affected substratebinding affinity. By contrast, the Alamutation enhanced substrate and inhibitor binding by 20-fold. Themost inactive of the mutants, Glu238, had a kcat/Km 4 ×10-6 lower than the wild-type value,suggesting that a positive charge on His 238is important for proper catalytic function. The Ala 238 mutant wasthe most active ADA, with akcat/Km2 × 10-3 lower than the wild-type value.NMR spectroscopy and crystallography revealed that thismutantis able to catalyze hydration of purine riboside, a ground-state analogof the reaction. These resultscollectively show that His 238 is not required for formation of thehydroxylate used in the deaminationand may instead have an important electrostatic role.
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